FR2488619A1 - PRODUCT CONTAINING IMMOBILIZED AMINOACYLASE AND PROCESS FOR PREPARING THE SAME - Google Patents

Abstract

PRODUCT CONTAINING IMMOBILIZED AMINOACYCLASE AND CONSISTING OF IMMOBILIZED AMINOACYCLASE AND A WATER-INSOLUBLE POROUS ANION EXCHANGER. THE AMINOACYCLASE IS ATTACHED TO THE POROUS ANION EXCHANGER AND CROSS-LINKED WITH A PROTEIN CROSS-LINKING AGENT, FOR EXAMPLE AN ALIPHATIC ALDEHYDE. CROSS-LINKING OF THE AMINOACYCLASE PREVENTS LOSS OF THE ENZYME BY RELEASE DURING ENZYMATIC REACTIONS IN WHICH THE ENZYME IS USED ON A SUPPORT.

Description

I 2488 6 19

  The present invention relates to a new

  product containing immobilized aminoacylase and a product

surrendered for its preparation.

  Aminoacylase is a known enzyme that hydrolyzes

  lysing the alpha- (N-acyl) -L-amino acids to the corresponding L-amino acids; this enzyme is therefore used for the optical resolution of DL-amino acids and in

similar applications.

  Recently, and with the aim of achieving a

  continuous use of an enzymatic reaction

  To prevent aminoacylase, attempts were made to immobilize this enzyme. For example, processes have been described in

  which the aminoacylase is adsorbed on an insoluble support

  luble in water or conjugated with an insoluble support

  in water by ionic bonds, covalent bonds.

  slow or gel conjugation; products containing the immobilized aminoacylase are thus obtained. Among these methods, and because of the ease of embodiment, the immobilization of aminoacylase by

  ionic bonds; however, this process has

  the enzyme may escape from a support when the concentration of a substrate to be treated or the ionic strength of a reaction system is high; as a result, the product containing the immobilized enzyme is

  stable; in addition, when a reaction is

  on a column filled with the product containing the enzyme

  immobilized me, one notes a strong loss of load.

  For example, it has been proposed to prepare the immobilized enzyme-containing product using an anion exchange resin or porous anion exchange resin as a water-insoluble carrier (Japanese Patent Application Publication No. 8.835 / 1973 and 16954 /

1976).

  These classic techniques are still far from

  to overcome the disadvantage of the immobilisation of amino-

  acylase by ionic bonds, ie that the enzyme

  is likely to escape from the support when the concentration

  treatment of a substrate to be treated or the ionic strength of a

reaction system is strong.

  Following extensive research, the De-

  The applicant found that a product containing the aminoacyla * was immobilized by fixing the latter on a water insoluble porous anion exchanger (abbreviated hereinafter as porous anion exchanger) and treating the product obtained. by a protein crosslinking agent; it then found that the product obtained did not

  not the disadvantages described above.

  The present invention therefore aims at the

  development of a new product containing aminoac-

  immobilized and which offers advantages, in parti-

  the almost total absence of leakage of the carrier-borne enzyme, even when treating a high-concentration substrate or when operating in a high-ionic reaction system, and in addition a low pressure drop in the process of reaction. The invention also comprises a process for preparing the product containing

Immobilized aminoacylase.

  Other purposes and advantages of the invention are

  will read the following description.

  These goals and benefits have been achieved in a

  product containing the immobilized aminoacylase and which

  takes this enzyme and a porous anion exchanger, the

  noacylase being attached to the porous anion exchanger and

  ticulated with a protein crosslinking agent. The

  containing the immobilized aminoacylase according to

  vention is prepared by attaching the enzyme to an

  Porous aneurysm and subsequent treatment of the product

  obtained by a protein crosslinking agent.

  In the present invention, the origin of the amino

  acylase to be fixed on the porous anion exchanger

  does not kill a critical factor and we can use an amino-

  acylase, provided that it is capable of

  lyse an N-acyl-L-amino acid into the corresponding L-amino acid

  ing. Preferably, an arminoacylase with high enzymatic activity will be used; produced by fungi belonging to

  to the genera Aspergillus, Penicillium and Mucor. The aminoacy-

  The method to be used can be obtained from such a source by any technique.

  The porous anion exchanger used in the

  vention is a porous material insoluble in water

  appropriate particle size, presenting a large

  number of pore sizes ranging from several tens to several hundreds of nm and a large specific surface area and a large pore volume, into which

  anion exchange groups. It is better to

  use as porous anion exchanger a phenolic resin

  as porous (for example a phenol-formaldehyde condensate), a porous styrene resin (for example a styrene-divinylbenzene copolymer), a porous silica or a porous glass with an average particle size of about 0.1 to 1, 2 mm, more especially 0.3 to 0.8 mm, with a pore size of about 15 to 300 nm, a specific surface area of about 10 to 150 m 2 / g and a pore volume of about

  0.3 to 1 ml / g, in which an exchange group was introduced.

  weakly basic anion group such as an amino group

  primary, secondary or tertiary, for example amino,

  thylamino or dimethylamino; or a group exchanger

  strongly basic anions such as a quaternary ammonium group

  ternary, for example trimethylammonium or dimethylhydro-

  xyéthylammonium. Examples of anion exchangers

  the most preferred are a porous silica with

  methylimmonium, with a pore size of about 100 nm, pore volume: about 0.8 ml / g, specific surface area:

  about 25 m 2 / g and particle size 100 to 200 m

  crons (product of the firm RhUne-Poulenc Industries,

  trade mark "Spherosil QMA"); a styrene copolymer

  porous ne-divinylbenzene containing trimethyl-

  ammonium, at a pore size of about 45 to 50 nm, pore volume: about 0.96 ml / g, specific surface area: about 30 m 2 / g and particle size of 0.35 to 0.55 mm (product of Mitsubishi Chemical Industries Ltd.,

  trademark "Diaion HPA-25"); polystyrene

  to a trimethylammonium group having a pore size of about 25 nm, a pore volume of about 0.35 ml / g, a surface area of about 25 to 35 m 2 / g and a particle size of 0.3 at 1.2 mm (product of the firm

  Diamond Shamrock Corporation, trademark "Duo

  lite A-161 ") a porous polystyrene with dimethylhydroxy groups

  droxyethylammonium, pore size: about 25 nm,

  pore volume: about 0.35 ml / g, specific surface area: about 25 to 35 m² / g and particle size: about 0.3 to 1.2 mm (product of Diamond Shamrock Corporation,

  trademark "Duolite A-162"); a phenolic resin

  porous phenolic hydroxyl group, pore size: about 15 nm, pore volume: about 0.7 ml / g, specific surface area: about 80 to 120 m 2 / g and

  particle: about 0.3 to 1.2 mm (product of the firm Dia-

  mond Shamrock Corporation sold under the trademark "Duolite A-

  561 "), a porous styrene-divinylbenzene copolymer with a

  trimethylammonium pes having a pore size of

  30 nm, a pore volume of about 0.5 ml / g, a surface area of about 25 m2 / g and a

  particle size of 0.3 to 0.8 mm (product of Dow Che-

  mical, trademark "Dowex MSA-1"); a polystyrene

  porous ring containing trimethylammonium groups having a di-

  pore size of about 30 to 200 nm, a volume of

  of about 0.95 ml / g, a surface area of about 42 m 2 / g and a particle size of about 0.4 to 0.5

  mm (a product of Rohm & Haas Co., a trademark of

  "Amberlite IRA-904") and similar products.

  Examples of crosslinking agents of

  C 2 -C 6 aliphatic dialdehydes, such as glyoxal,

  malonic di-aldehyde, succinic dialdehyde, aldehyde

  glutaric acid, adipic aldehyde and aldehydes

  gists. Glyoxal and glutaraldehyde are preferred.

2488619

  To prepare the product containing the aminoacylase

  immobilized according to the invention, the first and foremost

  minoacylase on the porous anion exchanger. This operation

  is preferably carried out by contacting the enzyme with

  with the porous anion exchanger in water or a solution

  appropriate buffer. It is possible to use the aminoacylase

  te or purified. Amount of aminoacylase to be used

  hangs from the particular porous anion exchanger we put

  but in general, it is preferable to use

  Viron 100 to 10,000 units and more especially 1,000 to 5,000 units of aminoacylase per ml of the exchanger. Among

  buffer solutions that are suitable, we will mention a solution

  phosphate buffer and a borated buffer solution and more especially a buffer 0.05 to 0.1 M, pH: about 7 to

9.

  The porous anion exchanger on which we have

  the aminoaxylase is then treated with the crosslinking agent

  proteins. This operation is preferably performed

  killed by contact of the exchanger with the crosslinking agent

  in water or buffer solution as described above.

  extra. In this operation, if the concentration of the crosslinking agent is too low, the crosslinking is insufficient.

  and it is difficult to prevent an escape of

  zyme. On the other hand, if the concentration of the crosslinking agent is too high, the enzyme is deactivated. Therefore, the amount of crosslinking agent should be adjusted according to the amount of enzyme adsorbed on the porous anion exchanger; in general, the crosslinking agent is preferably used for

  a final concentration of 0.01 to 0.1% by volume

to the reaction mixture.

  It is also possible to prepare the product containing

  immobilized aminoacylase by contacting the

  the aminoacylase and the cross-linking agent with the

  porous anions in water or a buffer solution such as

  as described above. This procedure is more

  mode as the multi-stage reaction described above

  because the desired product containing the immobilized aminoacylase

  can be obtained in one container and in one

more simple operation.

  In both operations, it is preferable to fix the aminoacylase on the porous anion sample and to treat with the protein crosslinking agent at a temperature of about 4 to 100 ° C. in order to limit as much as possible the deactivation of the enzyme and improve 1 '

  absorption and crosslinking efficiency.

  The preparation of the product containing the aminoacid-

  immobilized can be carried out batchwise or

  continuous on a column. When operating in discontinuous, -

  the aminoacylase is dissolved in water or a solution of

  in which the porous anion exchanger is then added. The mixture is stirred under the appropriate conditions

  for fixing, as described above, and ob-

  therefore holds a product containing the enzyme attached to the sample

  geur. After separating this product from the reaction mixture

  by a known technique such as decantation,

  filtration or centrifugation, the

  in a solution of the protein crosslinking agent in water or a buffer solution and stirred in

  conditions appropriate for crosslinking, such as

  written above; the desired product containing the immobilized aminoacylase is thus obtained. In the variant,

  when the crosslinking agent of proteins is added directly

  to the reaction mixture containing the heat exchanger

  on which the enzyme has been fixed and the

  under the same conditions as above, one can par-

  to the desired product, containing the immature aminoacylase

  bilized, in a single container. The product obtained,

  holding the immobilized aminoacylase can be easily separated

  reaction mixture by a known technique as described above. When continuously preparing on a column, water or a buffer solution containing the aminoacylase is passed over a packed column of the porous anion exchanger; we then do

  pass on the water column or buffer solution con-

  holding the crosslinking agent of proteins; we thus obtain

  the desired product containing the immobilized aminoacylase.

  Preferably, the two solutions, aminoacylase and protein crosslinking agent, will be passed into the

  column at a VS of about 0.1 to 0.5.

  When using the amino-containing product

  immobilized acylase according to the invention in a reaction

  zymatic, the enzyme hardly escapes from the

  porous anion changer, even when the substrate to be treated is in high concentration and when the reaction system has a high ionic strength; as a result, the product

  containing the immobilized aminoacylase remains stable-and

  serve an excellent enzymatic activity during

have been prolonged.

  The following examples illustrate the invention

  without limiting it; in these examples, the indica-

  Parts and% are by weight unless otherwise stated

opposite.

Example 1

  Various products containing the aminoacid-

  immobilized as described below. We react

  each of the products with a substrate solution in

  tinu and after a while the report

  residual enzyme activity and enzymatic activity

initial tick.

  1. Preparation of products containing amino

immobilized acylase

  (i) product containing the immobilizing aminoacylase

according to the invention.

  Each of the heat exchangers is inflated in the water.

  porous ions of Table I below; to 10 ml of exchanger

  swollen, add a solution of crude aminoacylase obtained

  nude from Aspergillus oryzae (50 ml) Prepared by

  dissolution of the crude aminoacylase (500 mg, 15,000 micro-

24884-1 9

  moles / h) in 0.05 M phosphate buffer (pH 7.5, 50 ml).

  To the resulting mixture, glutaraldehyde is added in an amount such that the final concentration of this reagent in the mixture is 0.05% by volume. After 18 hours of shaking at 10 C, the reaction mixture is filtered and

  the residue is washed with water; we obtain the product containing

immobilized aminoacylase.

  (ii) Comparative products (1) containing amino-

immobilized cylase.

  Each of the heat exchangers is inflated in the water.

  porous ions of Table I below; to 10 ml of the swollen exchanger is added a solution of crude aminoacylase obtained from Aspergillus oryzae (50 ml) prepared as described in (i) above. After 18 hours of shaking at 10 ° C., the reaction mixture is filtered and the residue is washed at

  the water; the product containing the aminoacylase im-

mobilized. -

  (iii) Comparative product (2) containing the amino

immobilized acylase.

  The same procedure is observed as in (i) above

  above, but replace the porous anion exchanger with

  an anion exchange resin of the gel type; we then obtain

  if a product containing the immobilized aminoacylase.

2. Application.

  One column of each of the products

  holding the immobilized aminoacylase obtained above (10 ml).

  A 0.6 M aqueous solution of Nacetyl-DL-methionine (pH 7.0, containing 5 × 10 ') is passed through the column at 37 ° C.

  moles of Co ++), thereby causing an enzymatic reaction.

  To measure the initial enzymatic activity of the product, the substrate solution is passed over the column at

  flow rate of 80 ml / h and the amount of L-methioni-

  not contained in the eluate. The activity is expressed as the amount in micromoles of L-methionine formed by h. After measuring the initial enzymatic activity of the product, the enzymatic reaction is continued for 10 days and the substrate solution is passed through the column at a rate of

  6 ml / h. After 10 days, enzymatic activity is measured

  residue of the product by the same procedure as for the initial activity. The relationship between the residual activity and the initial activity is expressed by attributing

  An arbitrary value of 100 to the initial activity.

3. Results.

  The results obtained are reported in the table.

  I below. The examination of these results clearly shows

  that the product according to the invention containing the aminoacid-

  immobilized enzyme has a residual enzyme activity much higher than that of the comparative products and

  its high enzymatic activity for prolonged periods.

  On the other hand, the enzymatic activity of the comparative product 1

  decreases over time. In addition, in the comparative product

  tif 1, we observe a support even in that. In the case of the enzyme that did not escape the enzyme with the

  initial stage of the enzymatic reaction

  comparative product 2, we can

  not adsorbed at all on the resin.

Table I

  Product Heat exchanger Treatment Activity of the aminoacy of anions by immobilized lase porous * crosslinking mm ilise initial after __ _____10 days

A 100 107

  According to B 100 88 the invention C yes 100 86 tion D 100 90

E 100 102

F 100 103

  Compared with 100 52 tif (1) C no 100 47

F 100 37

G control no 0 -

  (2) +: Symbols A to G of Table I refer to the following porous anion exchangers:

  A. porous polystyrene with trimethylamine groups

  nium, pore size: about 25 nm, pore volume: about 0.35 ml / g, specific surface area: about 25 to 35 m 2 / g, particle size: about 0.3 to 1.2 mm (product of Trade Duolite.-161 Diamond Shamrock Corporation). B. porous phenolic phenolic phenolic resin, pore size: about 15 nm, pore volume: about 0.7 ml / g, specific surface area: about 80 to 120 m 2 / g and particle size: about 0.3 to 1.2 mm (Diamond brand product Duolite A-561)

Shamrock Corporation).

  C. porous styrene-divinylbenzene copolymer with trimethylammonium groups, pore size: approximately

  mm, pore volume: about 0.5 ml / g, specific surface area

  about 25 m2 / g and particle size: approx.

  0.3 to 0.8 mm (Dowex MSA-1 commercial product from

Dow Chemical Co.).

D. Porous trimethylammonium porous styrene-divinylbenzene copolymer, pore size: about 50 nm, pore volume: about 0.96 ml / g, specific surface area: about 30 m 2 / g and particle size: about 0.35 0.55 mm (commercial product Diaion HPA-25

  from Mitsubishi Chemical Industries, Ltd.).

  E. porous polystyrene with trimethylmethylene groups

  nium, pore size: about 30 to 200 nm, pore volume: about 0.95 ml / g, specific surface area: about 42 m2 / g and particle size: about 0.4 to 0.5 mm (commercial product Amberlite IRA-904 from the firm Rohm and

Haas Co.).

  F. Porous silica with trimethylammonium groups,

  pore size: about 10 nm, pore volume: about

  0.8 ml / g, specific surface area: about 25 m 2 / g,

  particle size: 100 to 200 microns (commercially available product Spherosil QOA from RhOne Poulenc Industries and G. trimethylammonium gel type anion exchange polystyrene resin, conventional commercial product Duolite A-101D Diamond Shamrock (Corporation).

  **: the activity is expressed in relation to the initial activity

counted-for 100.

Example 2

  Operating as described in Example 1 (i) and

  (ii), the product containing the immature aminoacylase is prepared

  bilized according to the invention and the comparative product using

  porous silica containing trimethylammonium groups,

  pore size: about 100 nm, pore volume: approx.

  0.8 m / g, specific surface area: about 25 m 2 / g,

  particle size: about 1,000 to 2,000 microns (product

  of the trade Spherosil OMA of the firm Rhone Poulenc Indus-

tries).

  Each of the products containing the immature aminoacylase

  bilized is subjected to continuous enzymatic reactions on N-acetyl-1-phenylalanine (pH 7, containing 5 x 1074 moles of Co ++) and N-acetyl-L-methtonine (pH 7, containing x 10 -4 moles of Co ++) as respective substrates.

  The results obtained are reported in Table II below.

  after. Examination of these results clearly shows that the enzymatic activity of the product containing the aminoacylase

  immobilized, according to the invention remains stable during

  have been prolonged. In the comparative product, since the enzyme has escaped from the reaction system even in the initial stages of the reaction because of the strong

  concentration of the substrate, the enzymatic activity of the

  duit is weak and decreases again with the progress of the reaction.

  It is therefore clear that the product containing the amino.

  immobilized acylase according to the invention is capable of

  to demonstrate superior efficiency even with a substrate

12 2488619

high concentration.

Table II

  *: the activity is expressed in micromoles of L-amino acid

  formed by h and per 10 ml of the product containing the

  immobilized minoacylase (the epitope will be expressed

the same way below).

Example 3

  The aminoacylase is immobilized on the same sample

  of porous anions than in Example 2, by adding to this exchanger a solution of crude aminoacylase obtained at

  Aspergillus oryzae (50 ml) -prepared by dissolving

  crude aminoacylase solution (500 mg, 15,000 micromol / hr) in a 0.05 M phosphate buffer solution, pH 7.5_7

  then adding glutaraldehyde to the concentration

  indicated in Table III and concluding as

written in example 1.

  The various products obtained, containing amino-

  immobilized acylase but treated with different concentrations

  glutaric aldehyde, are subject to the reaction

  enzymatic process with N-acetyl-DL- as substrate

  methionine at 0.6 mole / liter, in order to compare their

  bilities. The results obtained are reported in the

Table III below.

  Product Concentrate Substrate- Activity of amino-

  immobilized acylase * substrate, moles / 1 initial After ______ ____ _ _ __ 20 days

According to N-acetyl-DL-

  According to N-Acetyl LD 0.6,600 6,900 methionine 1,

ven- N-acetyl-DL-

  phenyl-ala- 0,4,600 5,400 nine

N-acetyl-DL

  com- methionine 0.6 4.300 1.600

para- N-acetyl-DL-

  The results reported in this table show that when the glutaric aldehyde concentration is low, the enzyme escapes from the reaction system in an initial stage thereof. because of insufficient crosslinking. On the other hand, when the concentration of glutaraldehyde is high, the enzymatic activity is

  decreased. This results in an undesirable decrease in

tivity of the product.

Table 3

  Product Activity Concentration of Aminoacid-

  glutaric aldehyde, immobilized lase% in initial volume After days

0.005 5.400 4.300

  According to 0,01 8,700 8,500 the inven- 0,05 6,500 6,900 tion 0,1 5,900 6,900 Compara- - 4,300 1,600 tif

Example 4

  Porous silica containing trimethylammonium groups is inflated in water, pore size approximately

  nm, pore volume: about 0.8 ml / g, surface area

  about 25 m2 / g, particle size: approx.

  100 to 200 microns (commercial product Spherosil QMA

  from Rhone Poulenc Industries). To 10 ml of the silicone

  this porous swollen, we add an aminoacylase solution

  raw material obtained from Aspergillus oryzae (50 ml)

  prepared by dissolving crude aminoacylase (500 mg, 1,500 micromol / h) in a 0.05M phosphate buffer solution,

  pH 7.57. To the resulting mixture, gluteal aldehyde is added.

  in an amount corresponding to a final concentration of 0.05% by volume in the mixture. After 18 hours of shaking at 10 C, the reaction mixture is filtered,

  wash the residue with water; 10 ml of the product is obtained.

holding the immobilized aminoacylase.

  This product has an aminoacylase activity

than 6,500 micromoles / h.

Example 5

  By operating as described in Example 4, a product containing the immobilized aminoacylase is obtained.

  from the porous styrene-divinylbenzene copolymer con-

  containing trimethylammonium groups, pore size: about 45 to 50 nm, pore volume: about 0.96 ml / g,

  surface area: about 30 m2 / g and dimension of

  ticle: about 0.35 to 0.55 mm (commercial product Diaion HPA-25 from Mitsubishi Chemical Industries Ltd).

  10 ml of a product containing the amino

  immobilized acylase which has aminocylation activity

of 5,300 micromol / h.

Example 6

  By operating as described in Example 4, a product containing the immobilized aminoacylase from porous polystyrene containing trimethylammonium groups is obtained.

  having a pore size of about 25 nm, a volume of

  pore size of about 0.35 ml / g, a specific surface area

  from about 25 to 35 m 2 / g and a particle size of

  0.3 to 1.2 mm (Trade product Duolite A-161 from

Shamrock Corporation).

  10 ml of a product containing the amino

  immobilized acylase which has aminocylation activity

of 2,600 micromol / h.

Example 7

  By following the procedure of Example 4, there is obtained a product containing the immobilized aminoacylase from a porous styrene-divinylbenzene copolymer with trimethylammonium groups, pore size: approximately

  nm, pore volume: about 0.5 ml / g, surface area

  of about 25 m2 / g and particle size: approx.

  0.3 to 0.8 mm (Dowex MSA-1 commercial product from

Dow Chemical Co.).

  10 ml of a product containing the amino

  immobilized acylase and whose aminoacylase activity is

of 2,200 micromoles / h.

Example 8

  By following the procedure of Example 4, a product containing the immobilized aminoacylase from a porous styrene-divinylbenzene copolymer containing trimethylammonium groups, pore size approximately

  at 200 nm, pore volume: about 0.95 ml / g, over

  specific surface area: about 42 m2 / g and particle size

  the: about 0.4 to 0.5 mm (commercial product Amberlite

  IRA-904 from Rhom & Haas Co.).

  10 ml of a product containing the amino

  immobilized acylase and whose aminoacylase activity is

of 4,600 micromoles / h.

RE: ENDICATIONS

  1) A product containing immobilized aminoacylase

  , consisting of aminoacylase and an anion exchanger

  porous insoluble in water, characterized in that the amino

  acylase is attached to the porous and crosslinked anion exchanger

  by a protein crosslinking agent.

  2) Product according to claim 1, characterized in that the porous anionic water-insoluble exchanger has a pore size of about 15 to 300 nm, 10. a pore volume of about 0.5 to 1.0 ml / g, a specific surface area of about 10 to 150 m 2 / g and a

particle of about 0.1 to 1.2 mm.

  3) Product according to claim 1 or 2, characterized

  in that the porous anion exchanger insoluble in

  the water is chosen from the group consisting of a phenolic resin

  porous, porous styrene resin, silica

  porous glass and a porous glass, all of which have a

anion.

  4) Product according to claim 3, characterized in that the anion exchange group is selected from primary amino groups, secondary amino groups, tertiary amino groups, quaternary ammonium groups

  and phenolic hydroxy groups.

  ) Product according to claim 3 or 4, characterized

  in that the porous styrene resin is a copolymer

  porous styrene-divinyl-benzene mother carrying a group

anion changer.

  6) Product according to claim 3 or 4, characterized

  in that the porous phenolic resin is a porous phenol formaldehyde condensate having an anion exchange group. 7) Product according to claim 1, characterized

  in that the crosslinking agent of proteins is a diald6-

C2-C6 aliphatic hydroxide.

  Product according to claim 7, characterized

8)

  in that the aliphatic dialdehyde is selected from the group consisting of g] yoxal, malonic dialdehyde, succinic dialdehyde, glutaraldehyde and adipic aldehyde. 9) Product according to claim 8, characterized in

  what the aliphatic dialdehyde is glyoxal or aldehyde

glutaric. -

  ) Process for the preparation of the product containing

  immobilized aminoacylase according to any one of

  Claims 1 to 9, which method is characterized in that

  the aminoacylase is attached to a porous anion exchanger.

  soluble in water and treated the product obtained by a

crosslinking agent of proteins.

  11) Method according to claim 10, characterized

  in that the water-insoluble anion exchanger pre-

  a pore size of about 15 to 300 nm, a

  pore volume of about 0.3 to 1.0 ml / g, a specific surface area

  from about 10 to 150 m2 / g and a parti-

from about 0.1 to 1.2 mm.

  12) Process according to claim 11, characterized in that the water-insoluble porous anion exchanger is selected from the group consisting of a porous phenolic resin, a porous styrene resin, a porous silica

  and a porous glass all carrying an anion exchange group.

  13) Process according to claim 12, characterized in that the anion exchange group is selected from primary amino groups, secondary amino groups, tertiary amino groups, quaternary ammonium groups

  and phenolic hydroxy groups.

  14) Method according to claim 12 or 13, characterized

  in that the porous phenolic resin is a condensate

  porous phenol-formaldehyde sat with anion exchange group.

  Process according to claim 12 or 13, characterized

  in that the porous styrene resin is a copolymer

18 2488619

  porous styrene-divinylbenzene mother carrying an exchange group

anion.

  16) Method according to claim 10, characterized

  in that the protein crosslinking agent is a dialdehyde

  C2-C6 aliphatic hydroxide. 17) The method of claim 16, characterized in that the aliphatic dialdehyde is selected from the group consisting of glyoxal, malonic dialdehyde, dialdehyde auxuccine, glutaraldehyde and aldehyde

adipic.

  18) Method according to claim 17, characterized

  in that the aliphatic dialdehyde is glyoxal or al-

glutaric dehyde.

  19) Method according to claim 12, characterized in that, for the attachment of the aminoacylase to the water-insoluble anion exchanger, from about 100 to

  10,000 units of aminoacylase per ml of the exchanger.

  Method according to claim 11, characterized

  in that, for the crosslinking, the crosslinking agent is used

  protein at a concentration of about 0.01 to

  0.1% of the volume of the reaction mixture.